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US-12617819-B2 - Intermediate used for biologically active polypeptide and method for preparing same

US12617819B2US 12617819 B2US12617819 B2US 12617819B2US-12617819-B2

Abstract

The present invention relates to a novel intermediate used for physiologically active polypeptides and a method for preparing the same. The novel intermediate may be effectively used as an intermediate for the preparation of physiologically active polypeptide pharmaceuticals, and may be efficiently used for the preparation of high-quality pharmaceuticals by providing a polypeptide intermediate of high yield and high purity.

Inventors

  • Wonkyoung Choi
  • Nari Kim
  • Jonghwan PARK
  • Sungjun PARK
  • Namdu Kim
  • Youngbum Cho

Assignees

  • HANMI FINE CHEMICAL CO., LTD.

Dates

Publication Date
20260505
Application Date
20200214
Priority Date
20190215

Claims (17)

  1. 1 . A peptide intermediate compound represented by Chemical Formula 1 below: R-cyclo (Glu-Lys-Arg-Ala-Lys)-Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys-X (SEQ ID NO: 1): wherein, R is selected from the group consisting of H, linear or branched C 1-12 alkyl, linear or branched C 1-12 alkyloxycarbonyl, linear or branched C 2-12 alkenyl, C 3-10 cycloalkyl, heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl, and heteroaryl; X is selected from the group consisting of H, linear or branched C 1-12 alkyl, linear or branched C 1-12 alkyloxycarbonyl, linear or branched C 2-12 alkenyl, C 3-10 cycloalkyl, heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl, and heteroaryl; and these substituents may be further substituted with one or more identical or different substituents selected from the group consisting of H, halogen, cyano, linear or branched C 1-6 alkyl, linear or branched C 2-10 alkenyl, C 3-10 cycloalkyl, halo C 1-5 alkyl, hydroxy C 1-6 alkyl, amino, mono or di C 1-6 alkylamino, oxo, hydroxy, C 1-6 alkoxy, C 6-12 arylsulfonyl, and C 1-6 alkylsulfonyl.
  2. 2 . The peptide intermediate compound of claim 1 , wherein R is selected from the group consisting of H, linear or branched C 1-12 alkyloxy, C 6-12 aryloxy, and C 1-6 alkyl C 6-12 aryloxy.
  3. 3 . The peptide intermediate compound of claim 2 , wherein R is tert-butyloxy or 9-fluorenylmethyloxy.
  4. 4 . The peptide intermediate compound of claim 1 , wherein R is H, and X is H.
  5. 5 . A resin composite compound represented by Chemical Formula 3 below: wherein A to D are protecting groups; A to D are each independently selected from the group consisting of triphenylmethyl (Trt), tert-butyl (tBu), t-butyloxycarbonyl (Boc), and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf); R is selected from the group consisting of H, linear or branched C 1-12 alkyl, linear or branched C 1-12 alkyloxycarbonyl, linear or branched C 2-12 alkenyl, C 3-10 cycloalkyl, heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl, and heteroaryl; X′ is a resin; and these substituents may be further substituted with one or more identical or different substituents selected from the group consisting of H, halogen, cyano, linear or branched C 1-6 alkyl, linear or branched C 2-10 alkenyl, C 3-10 cycloalkyl, halo C 1-5 alkyl, hydroxy C 1-6 alkyl, amino, mono or di C 1-6 alkylamino, oxo, hydroxy, C 1-6 alkoxy, C 6-12 arylsulfonyl, and C 1-6 alkylsulfonyl.
  6. 6 . The resin composite compound of claim 5 , wherein a resin of the resin composite compound is a polystyrene (PS)-based resin or a polystyrene-polyethylene glycol copolymer (PS-PEG copolymer)-based resin.
  7. 7 . The resin composite compound of claim 5 , wherein the resin is a Rink amide resin.
  8. 8 . A method for preparing a resin composite compound represented by Chemical Formula 3 below, comprising the steps of: (1) swelling a resin in a polar aprotic solvent to give a deprotected resin; (2) coupling by adding an activated protected amino acid to the deprotected resin in a reactor; (3) repeating step (2) until a peptide comprising the amino acid sequence of SEQ ID NO: 1 is formed to give a synthesized peptide; (4) preparing a partially deprotected resin by reacting the synthesized peptide of step (3) with tetrakispalladium, N-methylaniline, and phenylsilane in a solvent; and (5) cyclizing the synthesized peptide obtained in step (4) by adding a coupling reagent in a polar aprotic solvent: wherein A to D are protecting groups; A to D are each independently selected from the group consisting of triphenylmethyl (Trt), tert-butyl (tBu), t-butyloxycarbonyl (Boc), and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf); R is selected from the group consisting of H, linear or branched C 1-12 alkyl, linear or branched C 1-12 alkyloxycarbonyl, linear or branched C 2-12 alkenyl, C 3-10 cycloalkyl, heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl, and heteroaryl; X′ is a resin; and these substituents may be further substituted with one or more identical or different substituents selected from the group consisting of H, halogen, cyano, linear or branched C 1-6 alkyl, linear or branched C 2-10 alkenyl, C 3-10 cycloalkyl, halo C 1-5 alkyl, hydroxy C 1-6 alkyl, amino, mono or di C 1-6 alkylamino, oxo, hydroxy, C 1-6 alkoxy, C 6-12 arylsulfonyl, and C 1-6 alkylsulfonyl.
  9. 9 . The method of claim 8 , wherein the polar aprotic solvent in step (1) and step (5) is selected from the group consisting of dimethylformamide, dimethylacetamide, and mixtures thereof.
  10. 10 . The method of claim 8 , wherein the solvent in step (4) is selected from the group consisting of dichloromethane, chloroform, and mixtures thereof.
  11. 11 . The method of claim 8 , wherein the coupling reagent in step (5) is selected from 1-hydroxy-1H-benzotriazole/1,3-diisopropylcarbodiimide or HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate)/N,N-diisopropylethylamine.
  12. 12 . A method for preparing a physiologically active polypeptide represented by Chemical Formula 2 below or a pharmaceutically acceptable salt thereof, comprising: (1) a reaction step of obtaining a peptide-resin composite by reacting the resin composite compound of Chemical Formula 3 below with amino acids to form a physiologically active polypeptide of Chemical Formula 2; and wherein A to D are protecting groups; A to D are each independently selected from the group consisting of triphenylmethyl (Trt), tert-butyl (tBu), t-butyloxycarbonyl (Boc), and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf); R is selected from the group consisting of H, linear or branched C 1-12 alkyl, linear or branched C 1-12 alkyloxycarbonyl, linear or branched C 2-12 alkenyl, C 3-10 cycloalkyl, heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl, and heteroaryl; X′ is a resin; and these substituents may be further substituted with one or more identical or different substituents selected from the group consisting of H, halogen, cyano, linear or branched C 1-6 alkyl, linear or branched C 2-10 alkenyl, C 3-10 cycloalkyl, halo C 1-5 alkyl, hydroxy C 1-6 alkyl, amino, mono or di C 1-6 alkylamino, oxo, hydroxy, C 1-6 alkoxy, C 6-12 arylsulfonyl, and C 1-6 alkylsulfonyl H-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-cyclo (Glu-Lys-Arg-Ala-Lys) Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys-NH 2 (SEQ ID NO: 2) (2) a cleavage step of cleaving the physiologically active polypeptide of Chemical Formula 2 from the peptide-resin composite.
  13. 13 . The method of claim 12 , wherein the reaction step comprises deprotecting the peptide-resin composite in a polar aprotic solvent, wherein the polar aprotic solvent is selected from the group consisting of dimethylformamide, dimethylacetamide, and mixtures thereof.
  14. 14 . The method of claim 12 , wherein the cleavage step is carried out in a medium containing a solution of trifluoroacetic acid, at least one scavenger, and dichloromethane.
  15. 15 . The method of claim 14 , wherein the scavenger is selected from the group consisting of triisopropylsilane, triethylsilane, phenol, anisole, thioanisole, water, ethanedithiol, 1-dodecanethiol, dithiothreitol, and indole.
  16. 16 . The method of claim 12 , wherein the pharmaceutically acceptable salt is trifluoroacetate or acetate.
  17. 17 . The method of claim 12 , wherein the resin composite compound of Chemical Formula 3 is a Rink amide resin composite compound.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a National Stage of International Application No. PCT/KR2020/002084 filed Feb. 14, 2020, claiming priority based on Korean Patent Application No. 10-2019-0017766 filed Feb. 15, 2019. INCORPORATION BY REFERENCE OF SEQUENCE LISTING The content of the electronically submitted sequence listing, file name: Q266743 SEQ_ST25.txt; size: 60,254 bytes; and date of creation: Sep. 15, 2025, filed herewith, is incorporated herein by reference in its entirety. TECHNICAL FIELD The present invention relates to a novel intermediate used for physiologically active polypeptides and a method for preparing the same. More specifically, the present invention relates to a method for preparing a physiologically active polypeptide in a safer and more effective manner, a novel polypeptide intermediate used therefor, and a method for preparing the same. BACKGROUND ART Diabetes-related diseases, including obesity and type 2 diabetes, are among the typical metabolic diseases developing in modern society, and are recognized as an important threat to health worldwide. Accordingly, economic costs are also increasing rapidly. Research has been conducted on the development of pharmaceuticals that can be used for the treatment of obesity and diabetes, but these have the disadvantages of showing fatal side effects or having insignificant effects on the treatment of obesity. Therefore, research that can solve the problems arising with conventional therapeutics is actively underway, and recently, much attention has been paid to glucagon derivatives. Glucagon is produced in the pancreas when the blood glucose levels fall due to reasons such as medications, diseases, deficiency in hormones or enzymes, etc. Glucagon sends a signal for glycogen breakdown in the liver to induce the release of glucose and increases blood glucose to a normal level. In addition to the effect of increasing the blood glucose levels, glucagon suppresses appetite and activates hormone-sensitive lipase of adipocytes to promote lipolysis, thereby showing an anti-obesity effect. Various studies related to glucagon are underway. In one example, Korean Laid-Open Publication No. 10-2017-0080521 discloses a triple agonist having activities to all of glucagon, GLP-1, and GIP receptors and uses thereof. Such a peptide may be composed of substitution, addition, deletion, modification, and combinations thereof in at least one or more amino acids in the native glucagon sequences, and more specifically, it discloses an isolated peptide including the amino acid sequence represented by the following General Formula 1: [General Formula 1](SEQ ID NO: 49) Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10- Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18- Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu- Xaa27-Xaa28-Xaa29-Xaa30-R1  (SEQ ID NO: 103 of the KR 10-2017-0080521). The peptide may be prepared by way of methods known in the art, for example, synthesis via an automatic peptide synthesizer, genetic manipulation technique, or any other methods, depending on the length of the peptide. In order to use the peptides prepared by way of these various production methods as pharmaceuticals, the quality of high purity, a yield suitable for commercialization, and a production process suitable for mass production are required. Therefore, there is a need for a novel polypeptide intermediate that enables the efficient production of such physiologically active polypeptides and an efficient production method capable of providing the same. Accordingly, the present inventors have completed the present invention by developing a novel polypeptide intermediate and a method for preparing the same, as a result of research on a method for preparing a physiologically active polypeptide more safely and efficiently. PRIOR ART LITERATURE Patent Literature Korean Laid-Open Publication No. 10-2017-0080521 (2017 Jul. 10), Triple agonist having activities to all of glucagon, GLP-1, and GIP receptors DISCLOSURE Technical Problem It is one object of the present invention to provide a novel polypeptide intermediate and a resin composite compound used for physiologically active polypeptides. It is another object of the present invention to provide a method for efficiently preparing the novel polypeptide intermediate and resin composite compound. It is still another object of the present invention to provide a method for efficiently preparing a physiologically active polypeptide used for the novel polypeptide intermediate. Technical Solution In order to achieve the above objects, one aspect of the present invention provides a novel polypeptide intermediate represented by Chemical Formula 1 below: R-cyclo(Glu-Lys-Arg-Ala-Lys)-Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys-X (SEQ ID NO: 1):wherein, R is selected from the group consisting of H, linear or branched C1-12 alkyl, linear or branched C1-12 alkyloxycarbonyl, linear or branched C2-12 alkenyl, C3-10 cycloalkyl, heterocycloalkyl,